Inhalation anthrax is a life-threatening disease that would result from the release of Bacillus anthracis spores into the environment. The economic impact of such an event, which would be secondary only to the resulting morbidity and mortality figures, would be in the billions. Although approved antibiotics are available for use against anthrax, they represent standard classes currently being used for other diseases. Resistant strains of B. anthracis exist for certain antimicrobials and development of resistant strains to all the available drugs is achievable with current molecular tools. There is only one new antimicrobial in late-stage development that is effective in preventing inhalation anthrax infections. The goal of this R01 proposal is to develop a new assemblage of antimicrobials for the treatment of inhalation anthrax that will inhibit a critical metabolic enzyme, dihydrofolate reductase (DHFR). The anthracis DHFR is inherently resistant to the only clinical drug for this target. Thus, if multiple-resistant B. anthracis were covertly created, DHFR would likely be unchanged and these new antimicrobials would be effective against those strains. Through an R21, several new parent compounds were identified that inhibit B. anthracis through selective activity against the bacterial DHFR (IC50s of 46-100 nM) but not human DHFR (IC50s > 25,000 nM). This approach will involve a team of scientists from diverse disciplines, including biochemistry, microbiology & structural biology, in silico technology, protein chemistry, molecular biology, medicinal chemistry, and animal models. The application is a specific response to a RFA request to develop therapeutics for NIAID Category A, B, and C priority pathogens. This application will involve the following Milestones: Milestone 1: Synthesize 4 more parent compounds designed from crystallographic working model and from previous studies; Milestone 2: Propose 25 suggested 1st generation compounds using in silico drug design methods; Milestone 3: Begin PK and MTD studies; Milestone 4: Synthesize and assay 8 selected compounds from 1st generation derivatives; Milestone 5: Complete MTD/PK studies; Milestone 6: Evaluate 1st generation lead compounds for efficacy in mouse anthrax model; Milestone 7: Identify 1-3 lead compounds for preclinical development. This multidisciplinary effort will involve both academic and privately owned research organizations participating in a comprehensive team-based plan that will facilitate the advancement of a promising therapeutic through the product development pathway. The public health relevance is the development of a therapeutic countermeasure that will be effective against multidrug-resistant strains of anthrax.